Monitoraggio chimico della

biodisponibilità dei metalli pesanti

Paola Adamo

Dipartimento di Agraria

Università di Napoli Federico II

paola.adamo@unina.it

SUMMER SCHOOL

Napoli, 8-10 luglio 2014

Outline

Introduction

Heavy metals in soil

Bioavailability

Chemical extractions

Experimental works

I metalli pesanti

Metalli con densità > 4.5 g cm-3

greatest concern for Pb, Cd, Hg, As

Essenziale o

benefico per:

Funzioni biochimiche

As Metabolismo di carboidrati e flavonoidi

Cd Sostituisce Zn in diatomee marine

Co Azotofissazione simbiotica

Cr Attivazione enzimi

Cu Attivazione enzimi, fotosintesi, metabolismo carboidrati e proteine

Mn Fotosintesi, sintesi acidi nucleici, cofattore di enzimi

Mo Azotofissazione, assorbimento e traslocazione Fe

Ni Traslocazione N, attivazione idrogenasi

Se Componente della glutatione perossidasi

Zn Sintesi clorofilla, attivazione enzimi, metab. carboidrati e proteine

Biochimica dei metalli pesanti

Potentially toxic metals or elements

ESSENTIAL elements: Also termed micronutrients required in low concentrations (< 0.1%), but at high concentrations they may be toxic for plants, animals, and humans (e.g. Cu, Fe, Mn, Mo, Zn…)

NONESSENTIAL elements: even low concentrations in the environment can cause toxicity to both plants and animals (e.g., Hg, Cd, Pb)

In soil: natural vs anthropic sources

soil parent material Al As Cr Cu Fe Mn Ni Pb V

volcanoes As Cd Cu

Hg Ni Pb Zn

anthropogenic sources As Cd Cu Hg Mo Ni Pb

Se V Zn

metals cycle

Be>2 mg/kg (DL 152/06)

Be>6,3 mg/kg (ISPRA-ARPAC, 2010)

Adverse effects are not necessarily only manifested in the environment when PTMs have an anthropogenic origin. Naturally high concentrations of some elements also cause toxicity and lead to natural adaptation of the biota to these high concentrations.

Cr, Ni in serpentine soils (Kelepertzis et al., 2013; Kelepertzis and Stathopoulou, 2013) As in groundwater in Bangladesh, India, China, Mexico, etc. (Mahimairaja et al., 2005) Se in seleniferous soils (Dhillon and Dhillon, 2003)

Esempi:

Alyssum bertolonii

PTEs contamination is a problem in Europe

in UE ~ 3,5 million sites are potentially contaminated

Thematic Strategy for Soil Protection (CEC, 2006)

Rattan L., SSSAJ, 71, 2007

Soil

Food

Feed

Fuel (Bio)

Fiber

Feed

Food

increased

anthropogenic

demands on soil

resources

The problem of pro capita cultivated land scarsity is exacerbated by the extent and severity of soil contamination

Food security is threatened by soil contamination

Contamination of soil with potentially toxic elements (PTEs) poses serious risks for biota and human health

Worldwide, numerous urban-industrial, but also agricultural, sites and soils need to be investigated to define environmental hazards and to propose new occupation plans and eventually remediation treatments

Cairo - Urban soil

Solofra – agricultural soil

Bagnoli – ILVA –industrial soil

THE NEED OF SPECIATION

total or “pseudototal” content of trace elements in soil might minimize the risks

Mobility and bioavailability

Risk/toxicity

Remediation techniques

Management

La stessa legislazione italiana attualmente vigente (DM 471/99; DL 152/06), che definisce

i limiti accettabili di contaminazione, i criteri, le procedure e le modalità di messa in

sicurezza, bonifica e ripristino dei siti inquinati, tiene conto solo del contenuto totale

Tabella 1. Allegato V, parte IV, DL 152/06

‘classical’ definition of speciation

‘According to IUPAC the terms ‘speciation’ and ‘chemical

species’ should be reserved for the forms of an element

defined as to isotopic composition, electronic or oxidation

state, and/or complex or molecular structure

‘broader’ definition of speciation

Speciation science seeks to characterise the various forms

or, at least, the main pools in which trace elements occur in

soil.

Ure & Davidson, Chem. Spec. Env., Blackwell 2002

The ‘classical’ definition, appropriate to speciation in solution samples, would exclude most speciation studies on solid materials, such as soils and sediments, more properly defined as fractionation studies.

Major forms of PTMs in soil

Form

biological availability

Chemical interactions

Mobility Toxicity potential

Cationi

Cu(II), Pb(II),

Ni(II), Al(III),

Hg(II), Mn(II),

Cr(III), etc.

Anioni

Arseniato,

Seleniato,

Cromato, Selenito,

Molibdato, etc.

Forme ossidate

Cu(II)

Cromato(VI)

Arseniato(V)

Hg(II)

Seleniato(VI)

Vanadio(IV)

Forme ridotte

Cu(I) Cr(III)

Arsenito(III) Hg(I)

Selenito(IV)

Vanadio(II)

Ritchie and Sposito, Speciation in soils, Blackwell, 2002

Cd 2+

, CdCl + , CdSO 4

0

Cr 3+

, CrOH 2+

, organic Cu 2+

Ni 2+

, NiSO 4 0 , organic

Pb 2+ , organic, PbSO 4

0

Zn 2+ , organic, ZnSO 4

0

pH 3.5-6.0

Mn 2+ , MnSO 4

0 , organic

Fe-OH species, organic

Cd 2+

, CdCl + , CdSO 4

0

Cr(OH) 4 -

Cu(OH) , +

CuCO 3 0 , organic

Ni 2+

, NiHCO 3 + , NiCO 3

0

Pb(OH) + , Pb(OH) 2

0 , PbHCO 3

+ ,

PbCO 3 0 , organic

Zn 2+

, organic, Zn(OH)

+ , Zn(OH) 2

0 , ZnHPO 4

0 ZnCO 3

0 ,

pH 6.0-8.5

Mn 2+

, MnSO 4 0 , MnCO 3

0

Fe-OH species, organic

Prevalent species of PTMs in soil solution

Plant uptake and element concentration in soil solution

Chen, Agr. Ecos. Env., 2009 Nolan et al., JEQ, 2005

In soil quantity and quality of soluble forms are constantly and rapidly

changing and not frequently well correlated with plant uptake

PLANT ROOTS

Microorganisms

Nutrients

Pollutants

As

Se Cu Se

P P

Pb

P

organic acids, sugars,

amino acids, lipids,

flavanoids, proteins,

carbohydrates, coumarins,

(phyto)siderophores

humic substances,

phyllosilicates,

variable-charge

minerals

Adsorbimento non specifico Adsorbimento specifico

PROCESSI DI ADSORBIMENTO

Valori di CSC e quantità di Zn adsorbite da diversi costituenti solidi del suolo

(Brummer et al., 1983)

CSC

(mol/g pH7)

Zn adsorbito

(mol/g)

Calcite - 0.44

Bentonite 450 44

Acidi umici 1700 842

Ossidi di Fe amorfi 160 1190

Ossidi di Al amorfi 50 1310

Ossidi di Mn amorfi 230 1540

0

100

200

300

400

500

600

0 1 2 3 4 5 6 7

So

rbed

Cu

, m

mo

l kg

-1

Equil. metal conc. (x 104 mol)

no ligand

phtalate

fulvate

citratecitrate

fulvate

phtalate

No ligand

McBride, Kluver, 1991

0

20

40

60

80

100

4 5 6 7 8 9 10C

d s

orb

ed

, %

pH

Cd

DFOB/Cd = 4

D

Cd

DFOB/Cd= 4

Neubauer et al., EJSS 2002

Cu

Inhibited sorption

DFOB=desferrioxamine B

Influence of organic molecules on the sorption of

Cu and Cd on montmorillonite

Promoted sorption

In: Violante et al., Metals & Metalloids in Soil Env., Wiley 2008

La quantità biodisponibile di un MPT può

essere definita come la frazione del suo

contenuto totale nel suolo che può essere

assorbita da un organismo biologico

(Krishnamurti, 2008).

Suddivisione degli elementi in funzione del valore

del potenziale ionico (carica/raggio)

Schematizzazione della relazione tra mobilità dei metalli pesanti e

pH in suoli minerali a tessitura tendenzialmente sabbiosa

Bioavailability depends on plant

Root growth, morphology and depth

Root hairs

Mycorrhizae maize

trifolium

Root exudation

and rhizosphere

pH changes

Indicators of bioavailability Bioavailability of PTMs in soils can be examined using chemical extraction and bioassay tests that determine a fraction of the PTMs is bioaccessible. Chemical extraction tests include single extraction and sequential fractionation. Bioassay involves plants, animals, and microorganisms.

Estrazioni chimiche singole per valutare la

fitodisponibilità dei MPT nel suolo

Plant available species

Extractant Elements

Soluble + exchangeable

0.1/0.01/0.05 M CaCl2 Cd, Pb, Zn

0.1 M NaNO3 Cd, Cu, Pb, Zn

1 M NH4NO3 Cd, Ni

1 M NH4OAc, pH 7 Mo, Ni, Pb, Zn

+ organically-bound, specifically adsorbed by oxides and clay minerals

1 M NH4OAc + 0.01 M EDTA, pH 7

Cu, Mn, Zn

0.5 M NH4OAc+ 0.02/0.05 M EDTA

Cd, Cu, Fe, Mn, Mo, Pb, Zn

0.005/0.5M DTPA+0.01M CaCl2 + 0.1M TEA pH 7.3

Cd, Cu, Fe, Mn, Ni, Zn

Di riferimento in Paesi Bassi, Svizzera e

Germania

Lakanen and Ervio, 1971

Linsday and Norwell, 1978

Plant available species

Extractant Elements

Exchangeable + carbonate

acetic acid 0.43 M

Cd, Cu, Cr, Ni, Pb, Zn

Exchangeable + organic complexes

0.05 M EDTA, pH 7

EU harmonised procedures Measurement and Testing Programme

Certified reference materials: BCR-700, BCR-483 e BCR-484 (valori indicativi anche per le quantità estratte con CaCl2 0.01 mol l-1,

NaNO3 0.1 mol l-1 e NH4NO3 0.1 mol l-1)

Rauret et al., 2001

Aspetti critici dell’uso delle estrazioni chimiche

singole per accertare la fitodisponibilità dei MPT

Devised for agricultural and nutritional purposes

Element, soil and plant specific

Validity of application in heavily polluted or strongly

disturbed soils

Industrial soil urban soil

Le quantità estratte sono indicative di contenuti potenzialmente disponibili alla pianta o lisciviabili più che di contenuti effettivamente biodisponibili.

In suoli trattati con fanghi di depurazione, nonostante l’incremento del contenuto totale ed EDTA-estraibile di MPT, è stato osservato un limitato trasporto di metalli alle parti aeree di piante sia selvatiche sia coltivate (Zea mays), indicando un potenziale rischio di lisciviazione dei metalli piuttosto che di traslocazione nei tessuti delle piante e/o nella catena alimentare (Zheljazkov e Warman, 2004; Kidd et al., 2007).

Analogamente, in un suolo pluricontaminato di Aznalcóllar (Siviglia, Spagna), sebbene la biodisponibilità (estraibilità con DPTA) dei MPT fosse elevata, la fitoestrazione dei metalli da parte di Brassica juncea era troppo limitata ai fini di un’efficiente bonifica del suolo (Clemente et al., 2005).

Non è semplice fare previsioni circa la biodisponibilità dei MPT. Le reazioni che avvengono nell’ambiente suolo, infatti, raramente sono all’equilibrio, condizione che invece si determina durante le estrazioni chimiche singole. Elevata capacità dei suoli di reintegrare il pool biodisponibile di MPT a una velocità vicina a quella di assorbimento dei MPT da parte delle piante.

TOTALE PRE

800 mg/kg

BIODISPONIBILE PRE

100 mg/kg

PIANTA

30 mg/kg

BIODISPONIBILE POST

100 mg/kg

Informazioni utili circa la distribuzione dei metalli tra i diversi costituenti del suolo, e circa i meccanismi responsabili della reintegrazione della frazione di MPT assorbita dalle piante, possono essere ottenute mediante l’uso di estrazioni chimiche sequenziali. Approccio in particolar modo interessante nell’ambito di indagini di bonifica mediante fitoestrazione, dove può fornire informazioni circa gli effetti della crescita delle piante su specie, forme e frazioni di MPT presenti nel suolo da bonificare.

Sequential chemical extractions

+ acqua, soluzioni saline, acidi deboli

Step 3

Step 4

+ agenti riducenti

+ agenti ossidanti

+ acidi forti

solubili, scambiabili, associati ai carbonati

occlusi da ossidi di Fe e Mn

associati alla sostanza organica, solfuri

Minerali primari e secondari

Soil

Sequential chemical extractions

Step 2

Step 1

Five-stage procedure of Tessier et al. (1979)

1 1M MgCl2 pH7 exchangeable

2 1M NaOAc pH5 carbonates

3 0.04 M NH2OH.HCl in Fe and Mn oxides 25% HOAc

4 0.02 M HNO3 + 30% H2O2 (pH2)

3.2M NH4OAc in 20% HNO3 organic matter

5 HF/HClO4 residual

Step extractant trace element species

Silveira et al., Chemosphere 64, 2006

In Oxisols, with high Fe and Mn oxides:

0.05M NH2OH/HCl, pH2

Poor crystalline Fe oxides (0.2M oxalic acid + 0.2M NH4 oxalate pH 3, dark)

6 M HCl

0% 20% 40% 60% 80% 100%

1

2

Silveira

Tessier Ex Carb Org Res

Fe-Mn

Mn oxides cryst Fe-ox

Krishnamurti et al., 1995; Krishnamurti and Naidu, 2000

0% 20% 40% 60% 80% 100%

1

2Tessier Ex Carb Org Res Fe-Mn oxides

Metal-organic complex

0.1M Na4P2O7 pH 10

Krishnamurti

Metal-organic-complex-bound Cd in soils of

southern Saskatchewan (Canada)

Cd-humate complexes Cd-fulvate complexes

1 CH3COOH 0.11 M Soluble, exchangeable, carbonates HOAc-extractable

2 NH2OH.HCl 0.5 M Mn and Fe oxides bound

pH 2 (0.1M, pH1,5) reducibile

3 H2O2/CH3COONH4 1 M Organic matter associated, pH 2 sulphides oxidizable

4 HCl/HNO3 primary and secondary

minerals residual

The EU procedures

Step reagent geochemical forms

Certified reference materials: CRM-601 and CRM-701

Sequential chemical extractions

USEFULNESS

Improved understanding of

PTEs behaviour in

environmental samples

Useful in assessing potential

risks from environmental

contaminants

Useful in assessing

effectiveness of reclamation

treatments

MAIN LIMITATIONS

Uncritical application and

operational nature ignored

Lack of selectivity

Re-adsorption and

redistribution

Unexpected element

associations

Kim and McBride, Environ. Pollut. 144, 2006

Selectivity of 2 and 3 EU-BCR extraction steps was tested reversing their extraction order

0 0,5 1 1,5 2 2,5 3 3,5 4

0 10 20 30 40 50 60 70 80 90

mg kg-1 sewage sludge-amended soil

Cd

Cu

HOAc

H2O2 pH2

NH2OH.HCl pH2

poor selectivity

CRITICISM: PRESENTATION OF DATA

• Use of bar charts Avoid data presented only as a % of total

A has the greatest potential for mobilization

A, B and C have the same concentrations in

step 1, 2 and 3

Adamo et al., Environ. Pollut. 1996; Water, Air, Soil Pollut. 2002

Speciation of Cu and Ni in Sudbury soils

Cu (11-1891 mg kg-1)

Ni (23-2149 mg kg-1)

30% 27%

16% 8%

26% 17%

71%

11%

HOAc Residual

Oxidizable Reducible

Complementary use of EU-BCR sequential extraction

and SEM/EDS provides a more realistic speciation

Adamo et al., 1996; 2002

CAMPO AMERICANO

Parco Omo: stoccaggio residui di produzione (loppe, scorie di fusione, residui carbone e minerali,…)

Parco minerali: stoccaggio minerali utilizzati per la produzione di ghisa e acciaio

Parco fossili: stoccaggio combustibili fossili (derivati del petrolio, carbone)

La classificazione del territorio in unità di paesaggio (antropico): una fase preliminare indispensabile al campionamento

Adamo et al., STOTEN, 2002

Metal contamination of soils from ILVA iron-steel industrial plant of Bagnoli (south Italy)

Chemical evidences of low metal mobility

62%

27%

12%

Co

100%

Cr

Riducible 22%

HOAc 4%

Cu Oxidizable 17%

Residual

83%

Ni

65% 9%

Metal speciation in soils devoted to stocking raw materials

3.2 mm

1 mm

0.7 mm

Cu Co Cr Zn

mg kg-1

243 299 64 1000

245 222 56 1012

Translocation of metals down the profile bound to iron fine particles

WDS

EDS

OM

Adamo et al., STOTEN, 2002

Forms of Fe and metals associated with iron phases analysed by oxalate and

dithionite–citrate sequential dissolution technique

Step

Extractant Fe form Reference

1 NH 4 - oxalate

at pH 3 amorphous Fe Scwerthmann

(1964)

2 Dithionite - citrate (DC)

crystalline Fe Holmgren (1967)

3 HNO 3 /HF residue

1

2

3

SPECIATION OF TRACE METALS IN THE SOIL CLAY FRACTION (< 2 m)

Fe

OXA

49% DC

26%

RES

25%

OXA

44%

DC

7%

RES

49%

Zn

In the clay fraction (< 2 m) metals associated

with amorphous and crystalline iron

OXA

23%

DC 49%

RES

28%

Cu

DC

56%

RES

44%

Ni

DC

56%

RES

42%

OXA

2%

Cr

NH 4 - oxalate

at pH 3 (OXA)

Dithionite -

citrate (DC)

Organic and inorganic colloid migration in porous

media may enhance the mobility along soil profile of

metal contaminants generally characterised by low

solubility and high adsorption affinities

Profilo pedologico di un suolo inondato

C2 (5-8 cm)

Apb (8-60 cm)

Bb (60~ cm)

C1 (0-5 cm)C2 (5-8 cm)

Apb (8-60 cm)

Bb (60~ cm)

C1 (0-5 cm)Campioni indisturbati

Cr

1% 4% 5%

90%

2% 12%

72%

14%

1% 5%

59% 35%

1%

7% 41% 51%

1% 9%

67% 23%

C

E1 E3 E2

SE

RES OXI RED HOAc

Sequential extractions on overfloowed soils

2 mm M.O. PPL

Analisi al SEM/WDS di sezioni sottili

C2 (5-8 cm)

Apb (8-60 cm)

Bb (60~ cm)

C1 (0-5 cm)C2 (5-8 cm)

Apb (8-60 cm)

Bb (60~ cm)

C1 (0-5 cm)

Cr Cu

mg kg-1

1638 61 105 4

Deposizioni superficiali di sedimenti fluviali a struttura

massiva

800 µm M.O. PPL

Accumulo di materiali fini nei pori

C2 (5-8 cm)

Apb (8-60 cm)

Bb (60~ cm)

C1 (0-5 cm)C2 (5-8 cm)

Apb (8-60 cm)

Bb (60~ cm)

C1 (0-5 cm)

Cr Cu

mg kg-1

273 61 243 44

In profondità accumulo laminare di materiali fini

400 µm M.O. PPL

C2 (5-8 cm)

Apb (8-60 cm)

Bb (60~ cm)

C1 (0-5 cm)C2 (5-8 cm)

Apb (8-60 cm)

Bb (60~ cm)

C1 (0-5 cm)

Cr Cu

mg kg-1

63 8 24 2

13 8 18 4

8.0 mm

1.0 mm 1.0 mm

1.7 mm

Sequence of interconnected events between primary laminar

aggregates and silt/clay illuviation in soil

The study area was the medium-sized city of Ancona on the east coast of central

Italy. 21 urban soils were sampled from public parks (Pk1-7), private gardens,

neglected and reshaped areas, flower beds on the side of busy roads (Tr1-10) and

nearby the port and an oil refinery (In1-4).

Latitude 43°37’0’’N

Longitude 13°31’0’’E

Altitude 0-110 m asl

Surface 123 km2

Inhabitants 101 848

Density 828 inh./km2

Soils as indicators of urban pollution: an example from central Italy

Adamo et al., UEP -2012

Potentially toxic metals (PTMs) - total content

Only Pb and Zn concentrations were above the Italian law limits, in 33% and 48%

of the examined sites, respectively. Cu exceeded the law limit only in Palombella

park. Vehicular traffic and industrial areas were the main pollution sources

enhancing PTMs content in soils. Metal contents in industrial and roadside soils

were higher than in soils from parks.

0

50

100

150

200

250

300

mg/kg fraction

mg/kg soil mg/kg fraction

mg/kg soil mg/kg fraction

mg/kg soil mg/kg fraction

mg/kg soil mg/kg fraction

mg/kg soil mg/kg fraction

mg/kg soil

Cu Zn Pb Ni Co Cr

<2 µm 2-10 µm

10-50 µm >50 µm

PTMs - Physical fractionation

The finest (< 2 m) soil particles concentrated high amounts of metal pollutants.

This indicates the finest particles, more often inhaled or ingested, are dangerous

carriers of contaminants. The accumulation in the fine fraction only clearly suggests

that the source is anthropogenic. A lithogenic derivation would in fact generate a

more even distribution among sizes or a second mode in the coarse fraction.

CONCLUDING REMARKS

guidelines for PTMs in soil have to consider not only

their total contents, but also mobility, availability and

toxicity of contaminants

harmonized analytical procedures and certified

reference materials increase the quality of data

bioavailability, as currently understood, is a concept

more meaningful to legislators than to scientists

much work still remain to be conducted to investigate

the nature of PTMs species in polluted sites, with

special attention to bioavailability, colloidal migration

and physical fractionation